Dendritic cells cross-present latency gene products from Epstein-Barr virus-transformed B cells and expand tumor-reactive CD8(+) killer T cells.

Dendritic cells (DCs) are not targets for infection by the transforming Epstein-Barr virus (EBV). To test if the adjuvant role of DCs could be harnessed against EBV latency genes by cross-presentation, DCs were allowed to process either autologous or human histocompatibility leukocyte antigen (HLA)-mismatched, transformed, B lymphocyte cell lines (LCLs) that had been subject to apoptotic or necrotic cell death. After phagocytosis of small numbers of either type of dead LCL, which lacked direct immune-stimulatory capacity, DCs could expand CD8(+) T cells capable of killing LCLs that were HLA matched to the DCs. Necrotic EBV-transformed, major histocompatibility complex (MHC) class I-negative LCLs, when presented by DCs, also could elicit responses to MHC class II-negative, EBV-transformed targets that were MHC class I matched to the DCs, confirming efficient cross-presentation of LCL antigens via MHC class I on the DC. Part of this EBV-specific CD8(+) T cell response, in both lytic and interferon gamma secretion assays, was specific for the EBV nuclear antigen (EBNA)3A and latent membrane protein (LMP)2 latency antigens that are known to be expressed at low levels in transformed cells. The induced CD8(+) T cells recognized targets at low doses, 1-10 nM, of peptide. Therefore, the capacity of DCs to cross-present antigens from dead cells extends to the expansion of high affinity T cells specific for viral latency antigens involved in cell transformation.

EBNA1-specific CD4+ T cells in healthy carriers of Epstein-Barr virus are primarily Th1 in function.

The Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) maintains the viral episome in all host cells infected with EBV. Recently, EBNA1 was found to be the main EBV latency antigen for CD4+ T cells and could be recognized in cultures from all donors tested. We now identify a polarized Th1 phenotype and obtain evidence for its presence in vivo. When T cells were stimulated with dendritic cells infected with vaccinia vectors expressing EBNA1, 18 of 19 donors secreted IFN-gamma, whereas only two of 19 secreted IL-4. Magnetic selection was then used to isolate cells from fresh blood based on EBNA1-induced cytokine production. Specific IFN-gamma CD4+ cell lines were established from six of six donors and IL-4 lines from three of six. Only the Th1 lines specifically lysed targets expressing three different sources of EBNA1 protein. When the IgG isotype of EBNA1 plasma Ab's was tested, most specific Ab's were IgG1 and of a high titer, confirming a Th1 response to EBNA1 in vivo. Ab's to other microbial antigens generally were not skewed toward IgG1. Given emerging evidence that Th1 CD4+ T cells have several critical roles in host defense to viral infection and tumors, we propose that EBNA1-specific CD4+ Th1 cells contribute to resistance to EBV and EBV-associated malignancies.

Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.

Critical cytoplasmic domains of human interleukin-9 receptor alpha chain in interleukin-9-mediated cell proliferation and signal transduction.

Interleukin-9 receptor (IL-9R) complex consists of a ligand-specific alpha chain and IL-2R gamma chain. In this study, two regions in the cytoplasmic domain of human IL-9Ralpha were found to be important for IL-9-mediated cell growth. A membrane-proximal region that contains the BOX1 consensus sequence is required for IL-9-induced cell proliferation and tyrosine phosphorylation of Janus kinases (JAKs). Deletion of this region or internal deletion of the BOX1 motif abrogated IL-9-induced cell proliferation and signal transduction. However, substitution of the Pro-X-Pro in the BOX1 motif with Ala-X-Ala failed to abolish IL-9-induced cell proliferation but decreased IL-9-mediated tyrosine phosphorylation of JAK kinases, insulin receptor substrate-2, and signal transducer and activator of transcription 3 (STAT3) and expression of c-myc and junB. Another important region is downstream of the BOX1 motif and contains a STAT3 binding motif YLPQ. Deletion of this region significantly impaired IL-9-induced cell growth, activation of JAK kinases, insulin receptor substrate-2, and STAT3 and expression of early response genes. A point mutation changing YLPQ into YLPA greatly reduced IL-9-induced activation of STAT3 and expression of c-myc but did not affect cell proliferation. These results suggest that cooperation or cross-talk of signaling molecules associated with different domains of IL-9Ralpha other than STAT3 is essential for IL-9-mediated cell growth.

Uncoupling of stem cell inhibition from monocyte chemoattraction in MIP-1alpha by mutagenesis of the proteoglycan binding site.

We have studied the role of proteoglycans in the function of Macrophage Inflammatory Protein-1 alpha (MIP-1alpha), a member of the proteoglycan binding chemokine family. Sequence and peptide analysis has identified a basic region within MIP-1alpha which appears to be the major determinant of proteoglycan binding and we have now produced a mutant of MIP-1alpha lacking the basic charges on two of the amino acids within this proteoglycan binding site. This mutant (Hep Mut) appears to have lost the ability to bind to proteoglycans. Bioassay of Hep Mut indicates that it has retained stem cell inhibitory properties but has a compromised activity as a monocyte chemoattractant, thus suggesting uncoupling of these two properties of MIP-1alpha. Receptor studies have indicated that the inactivity of Hep Mut on human monocytes correlates with its inability to bind to CCR1, a cloned human MIP-1alpha receptor. In addition, studies using proteoglycan deficient cells transfected with CCR1 have indicated that the proteoglycan binding site in MIP-1alpha is a site that is also involved in the docking of MIP-1alpha to the monocyte receptor. The site for interaction with the stem cell receptor must therefore be distinct, suggesting that MIP-1alpha utilizes different receptors for these two different biological processes.

Heterodimers of placenta growth factor/vascular endothelial growth factor. Endothelial activity, tumor cell expression, and high affinity binding to Flk-1/KDR.

Here we show that the Escherichia coli expressed monomers of placenta growth factor (PLGF)129 and vascular endothelial growth factor (VEGF)165 can be re-folded in vitro to form PLGF/VEGF heterodimers. The purified recombinant PLGF/VEGF heterodimers and VEGF homodimers have potent mitogenic and chemotactic effects on endothelial cells. However, PLGF/VEGF heterodimers display 20-50-fold less mitogenic activity than VEGF165 homodimers. In contrast, PLGF129 homodimers have little or no effect in these in vitro assays. We also demonstrate the presence of natural PLGF/VEGF heterodimers in the conditioned media of various human tumor cell lines. While PLGF/VEGF heterodimers bind with high affinity to a soluble Flk-1/KDR receptor, PLGF129 homodimers fail to bind to this receptor. Cross-linking of 125I-ligands to human umbilical vein endothelial cells reveals that PLGF/VEGF heterodimers and VEGF165 homodimers, but not PLGF129 homodimers, form complexes with membrane receptors. VEGF165 homodimers and PLGF/VEGF heterodimers stimulate tyrosine phosphorylation of a 220-kDa protein, the expected size for the KDR receptor in human umbilical vein endothelial cells, whereas PLGF129 homodimers are unable to induce tyrosine phosphorylation of this protein. These data indicate that PLGF may modulate VEGF-induced angiogenesis by the formation of PLGF/VEGF heterodimers in cells producing both factors.

Immunohistochemical detection of active transforming growth factor-beta in situ using engineered tissue.

The biological activity of transforming growth factor-beta 1 (TGF-beta) is governed by dissociation from its latent complex. Immunohistochemical discrimination of active and latent TGF-beta could provide insight into TGF-beta activation in physiological and pathological processes. However, evaluation of immunoreactivity specificity in situ has been hindered by the lack of tissue in which TGF-beta status is known. To provide in situ analysis of antibodies to differentiate between these functional forms, we used xenografts of human tumor cells modified by transfection to overexpress latent TGF-beta or constitutively active TGF-beta. This comparison revealed that, whereas most antibodies did not differentiate between TGF-beta activation status, the immunoreactivity of some antibodies was activation dependent. Two widely used peptide antibodies to the amino-terminus of TGF-beta, LC(1-30) and CC(1-30) showed marked preferential immunoreactivity with active TGF-beta versus latent TGF-beta in cryosections. However, in formalin-fixed, paraffin-embedded tissue, discrimination of active TGF-beta by CC(1-30) was lost and immunoreactivity was distinctly extracellular, as previously reported for this antibody. Similar processing-dependent extracellular localization was found with a neutralizing antibody raised to recombinant TGF-beta. Antigen retrieval recovered cell-associated immunoreactivity of both antibodies. Two antibodies to peptides 78-109 showed mild to moderate preferential immunoreactivity with active TGF-beta only in paraffin sections. LC(1-30) was the only antibody tested that discriminated active from latent TGF-beta in both frozen and paraffin-embedded tissue. Thus, in situ discrimination of active versus latent TGF-beta depends on both the antibody and tissue preparation. We propose that tissues engineered to express a specific form of a given protein provide a physiological setting in which to evaluate antibody reactivity with specific functional forms of a protein.

Interleukin-9 induces tyrosine phosphorylation of insulin receptor substrate-1 via JAK tyrosine kinases.

Interleukin (IL)-9 stimulates the proliferation of a variety of hematopoietic lineages through its interaction with a receptor of the cytokine receptor superfamily. In the studies presented here, we have begun to characterize the downstream signaling pathways activated by IL-9. In addition to the activation of JAK1 and JAK3 tyrosine kinases, IL-9, unlike most hematopoietic cytokines but similar to IL-4, induces the tyrosine phosphorylation of a 170-kDa protein that is related to the insulin receptor substrate-1 (IRS-1). We further demonstrate for the first time that IRS-1 is not only associated with JAK1 but also tyrosine phosphorylated and functionally involved in IL-9 signaling in TS1 lymphocytes transfected with the murine IRS-1 cDNA. Cotransfection studies and in vitro experiments directly demonstrate that JAK1, JAK2, or JAK3 is capable of tyrosine phosphorylating IRS-1, suggesting a functional role for these kinases in vivo. Lastly, we demonstrate that IL-9 induces the tyrosine phosphorylation of Stat3 and in this regard differs from IL-4, which triggers tyrosine phosphorylation of Stat6. Taken together, these results strongly suggest that IL-9 and IL-4 utilize common and unique signaling pathways via inducing the similar and distinct tyrosine-phosphorylated proteins.

Characterization of recombinant soluble human transforming growth factor-beta receptor type II (rhTGF-beta sRII).

Recombinant human transforming growth factor soluble receptor Type II (rhTGF-beta sRII) corresponding to the 159 amino acid extracellular domain of hTGF-beta RII has been expressed in insect cells using the baculovirus expression system or in a mouse myeloma cell line. N-terminal sequence analysis of the purified protein revealed the removal of the 23 amino acid signal peptide. In SDS-PAGE, the rhTGF-beta sRII resolves into multiple bands due to N-linked glycosylation. Recombinant hTGF-beta sRII is a TGF-beta antagonist and will inhibit the biological activities of TGF-beta 1, TGF-beta 3, and TGF-beta 5 on TGF-beta-responsive cell lines, such as murine HT-2 or human TF-1 with an ED50 of approximately 0.3 micrograms/mL. However, hTGF-beta sRII does not inhibit TGF-beta 2 bioactivities in these cell lines, suggesting that hTGF-beta RII has low affinity for TGF-beta 2. Polyclonal antibodies to hTGF-beta sRII have been produced in goats and purified on Protein-G affinity columns. This antibody can inhibit TGF-beta 1,2,3,5-dependent bioactivities on human cell lines such as TF-1. Additionally, this antibody has species cross-reactivity and will also inhibit TGF-beta-dependent bioactivities on murine cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Aggregation of the chemokine MIP-1 alpha is a dynamic and reversible phenomenon. Biochemical and biological analyses.

Macrophage inhibitory protein (MIP)-1 alpha is a potent inhibitor of hemopoietic stem cell proliferation and is a member of a family of pro-inflammatory mediators, the chemokine family. This molecule along with other members of the chemokine family exists as a peptide of 8 kDa but has a strong tendency for noncovalent extensive self-aggregation. As this aggregation may interfere with biological activity, we have produced nonaggregating variants of MIP-1 alpha which display a range of molecular sizes. The mutants, produced by sequential neutralization of carboxyl-terminal acidic residues, display native molecular masses representative of tetramers, dimers, and monomers. Intriguingly when these mutants are assessed in comparison with native MIP-1 alpha for bioactivity in vitro, they are seen to be equipotent in both stem cell assays and in monocyte shape-change assays, suggesting that there is no requirement for aggregation in either of these biological contexts. This indicates that the aggregated MIP-1 alpha and the aggregated mutants spontaneously disaggregate under assay conditions and ultimately function as monomers. We have further demonstrated the ability of MIP-1 alpha to disaggregate spontaneously in dilute solution by enzyme-linked immunosorbent assay analysis of fractions obtained from gel filtration of varying concentrations of MIP-1 alpha. The aggregation of MIP-1 alpha is therefore a dynamic and reversible phenomenon which has little, if any, impact on bioactivity in vitro.

Transforming growth factor-beta activation in irradiated murine mammary gland.

The biological activity of TGF-beta, an important modulator of cell proliferation and extracellular matrix formation, is governed by dissociation of mature TGF-beta from an inactive, latent TGF-beta complex in a process that is critical to its role in vivo. So far, it has not been possible to monitor activation in vivo since conventional immunohistochemical detection does not accurately discriminate latent versus active TGF-beta, nor have events associated with activation been defined well enough to serve as in situ markers of this process. We describe here a modified immunodetection method using differential antibody staining that allows the specific detection of active versus latent TGF-beta. Under these conditions, we report that an antibody raised to latency-associated peptide detects latent TGF-beta, and we demonstrate that LC(1-30) antibodies specifically recognize active TGF-beta 1 in tumor xenografts overproducing active TGF-beta 1, without cross-reactivity in tumors expressing similar levels of latent TGF-beta 1. We previously reported that TGF-beta immunoreactivity increases in murine mammary gland after whole-body 60Co-gamma radiation exposure. Using differential antibody staining we now show that radiation exposure specifically generates active TGF-beta 1. While latent TGF-beta 1 was widely distributed in unirradiated tissue, active TGF-beta 1 distribution was restricted. Active TGF-beta 1 increased significantly within 1 h of irradiation concomitant with decreased latent TGF-beta immunoreactivity. This rapid shift in immunoreactivity provides the first evidence for activation of TGF-beta in situ. This reciprocal pattern of expression persisted for 3 d and was accompanied by decreased recovery of latent TGF-beta 1 from irradiated tissue. Radiation-induced activation of TGF-beta may have profound implications for understanding tissue effects caused by radiation therapy.

Involvement of IL-6 signal transducer gp130 in IL-11-mediated signal transduction.

IL-11 is a novel cytokine with a variety of biofunctions which overlap with those of IL-6. We have previously identified IL-11 specific binding protein which is distinct from that of IL-6 in a number of cell lines. The similarities in biofunctions and differences in ligand binding proteins between IL-11 and IL-6 prompted us to investigate whether IL-11 shares common signal transduction mechanisms with IL-6. We have examined early signals triggered by IL-11 or IL-6 in a multifactor-dependent human erythroleukemic cell line, TF-1. The results showed that IL-11 and IL-6 can both stimulate cell proliferation, induce similar pattern of protein tyrosine phosphorylation, and activate the same proto-oncogene (junB) expression in TF-1 cells. These findings imply that IL-11 and IL-6 share similar early signaling events with the possibility of using the same signal transducer, gp130. We next tested whether IL-11 induced signaling can be inhibited by anti-gp130 antibodies which blocked IL-6-mediated functions. It was observed that anti-gp130 antibodies abolished cell proliferation, protein tyrosine phosphorylation, and junB gene expression elicited by IL-11 or IL-6 in TF-1 cells. The same antibodies, however, had no effect on granulocyte-macrophage colony stimulating factor or erythropoietin-induced TF-1 cell proliferation. Finally, anti-IL-6R antibody inhibited the ability of IL-6, but not IL-11, to transduce early signals in TF-1 cells. These results demonstrate that IL-11 and IL-6 utilize different ligand binding proteins, but share common signal transducer, gp130, in TF-1 cells.

Characterization of a receptor for macrophage inflammatory protein 1 alpha and related proteins on human and murine cells.

Macrophage inflammatory protein 1 alpha (MIP-1 alpha) is a potent stem cell inhibitor and a member of a large and expanding family of related cytokines. In an effort to understand the molecular basis of the activities of MIP-1 alpha, we have sought to characterize the cellular receptors for this molecule. Our results demonstrate the presence of abundant MIP-1 alpha receptors on both human and murine cells. The receptor on K562 cells can bind a range of members of the MIP-1 alpha family and may thus be a general MIP-1 alpha family receptor. Murine FDCPmix cells also bind a range of members of this peptide family, although the receptor(s) that they express appear somewhat more selective for peptides capable of displaying stem cell inhibitory properties. The human and murine receptors do not bind members of the related interleukin 8 family of peptides and are thus distinct from the recently cloned interleukin 8 receptor. We suggest that the receptor on the murine cell is a candidate for the receptor responsible for articulating stem cell inhibitory signals following MIP-1 alpha binding.

Binding and internalization of transforming growth factor-beta 1 by human hepatoma cells: evidence for receptor recycling.

Cellular processing of 125I-labeled transforming growth factor-beta 1 was investigated in the human hepatoma cell lines Hep G2 and Hep 3B. Binding of 125I-transforming growth factor-beta 1 to cell surface receptors was specific, saturable and calcium-independent. Both cell lines exhibited a single class of high-affinity (Kd = 2.2 x 10(-10) mol/L) binding sites (4.5 x 10(3) for the Hep G2 cell; 1.5 x 10(3) for the Hep 3B cell) for both human and porcine transforming growth factor-beta 1. Binding was temperature dependent, time dependent and pH dependent. Cell-bound 125I-transforming growth factor-beta 1 was removed by brief exposure to acidic medium (pH less than 4) but was converted into an acid-resistant state rapidly after shifting the cells to 37 degrees C. Spontaneous dissociation of bound ligand over a 6 hr period at 4 degrees C was less than 10%. Disuccinimidyl suberate was used to covalently label 125I-transforming growth factor-beta 1 to cell-surface binding sites. Labeling of the ligand/receptor complexes was inhibited by unlabeled transforming growth factor-beta 1 but was unaffected by other growth factors. The radiolabeled complexes showed approximate molecular weights of 280,000, 85,000 and 65,000 when run on reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cell-bound 125I-transforming growth factor-beta 1 was internalized and degraded at 37 degrees C, and the products were released into the medium as trichloroacetic acid-nonprecipitable radioactivity. The lysosomotropic base chloroquine and the carboxylic ionphore monensin inhibited degradation and release of 125I-labeled products from the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Zidovudine in the management of primary HIV-1 infection.

Eleven subjects who presented with a clinical illness characteristic of primary HIV-1 infection were treated with 1 g zidovudine daily for a median period of 56 days (range, 28-111 days). Primary HIV-1 infection was confirmed in each subject by seroconversion and virus isolation. The acute phase of the illness resolved a median of 4 days (range, 3-14 days) from commencement of zidovudine. Six subjects reported symptoms that may have been side-effects of zidovudine, the most common being nausea in four subjects and headache in two. Treatment was discontinued in one subject who had persistent headache and nausea. Haemoglobin, haematocrit and erythrocyte counts decreased and mean corpuscular volume increased significantly during the treatment. None of the subjects developed anaemia and none required dose modification or blood transfusion as a result of haematological side-effects. There were no significant differences in the granulocyte count or the lymphocyte count during any week of treatment when compared with baseline levels. There were no significant differences in T-cell subset numbers of the subjects during treatment compared with a group of historical controls. HIV-1 was isolated from several subjects during and after termination of zidovudine treatment. The results of this investigation indicate that zidovudine is a safe drug to administer to people with primary HIV-1 infection. There was no clear evidence, however, of any clinical benefit in terms of resolution of the acute illness and no indication that the treatment would prevent development of persistent infection.(ABSTRACT TRUNCATED AT 250 WORDS)

The transforming growth factor-beta system, a complex pattern of cross-reactive ligands and receptors.

A new homodimer form of transforming growth factor-beta (TGF-beta), TGF-beta 2, has been identified in porcine blood platelets. TGF-beta 2 is homologous to ordinary TGF-beta (TGF-beta 1), which is also present in platelets. TGF-beta 1.2, a heterodimer containing one TGF-beta 1 chain and one TGF-beta 2 chain, has also been isolated. TGF-beta 1 and TGF-beta 2 interact differently with a family of receptors in target cells. A 280 kd receptor displays high affinity for both TGF-beta 1 and TGF-beta 2. Occupancy of this receptor by TGF-beta 1 or TGF-beta 2 correlates with the ability of these TGF-beta s to inhibit cell proliferation. In contrast, 65 kd and 85 kd receptors have high affinity for TGF-beta 1 but lower affinity for TGF-beta 2. The existence of distinct forms of TGF-beta that interact differently with a family of TGF-beta receptors could provide flexibility to the regulation of tissue growth and differentiation by the TGF-beta system.

Aspergillus nidulans beta-tubulin genes are unusually divergent.

Aspergillus nidulans has two beta-tubulin genes: benA, which is involved in both vegetative growth and asexual sporulation, and tubC, which is involved mainly in asexual sporulation. Both genes have now been cloned and sequenced. benA encodes a polypeptide of 447 amino acids (aa) and tubC encodes one of 449 aa. The two polypeptides differ by 78 aa residues but the net charge for the two proteins remains the same. The divergence between the amino acid sequences of the Aspergillus beta-tubulins is greater than that for any other two beta-tubulins yet described in the same organism. The benA gene has eight introns and the tubC gene has five, all of which correspond in amino acid position to introns in benA. The positions of some of these introns are conserved in other beta-tubulin genes. The 5'-splice site, internal, and 3'-splice site consensus sequences are similar to those found in other fungal introns. The transcriptional start points for each gene have been determined using primer extension and/or S1 nuclease mapping. Neither the benA gene nor the tubC gene contains a TATA sequence in its 5'-flanking region. The tubC gene has two repeated upstream sequences which are not found in benA. The sites of polyadenylation have been determined for each gene using S1 nuclease mapping. Neither gene contains a polyadenylation signal, AATAAA, typical of other eukaryotic genes.

Thioredoxin, glutaredoxin, and thioredoxin reductase from cultured HeLa cells.

Thioredoxin and glutaredoxin may be important in regulating cell metabolism by mediating interchanges between sulfhydryl and disulfide groups. Components of the thioredoxin/glutaredoxin system from cultured HeLa cells have been partially purified and characterized by using Escherichia coli adenosine 3'-phosphate 5'-phosphosulfate reductase, a thioredoxin/glutaredoxin-dependent enzyme on the pathway of sulfate reduction, as an assay system. In HeLa cells, a NADPH-thioredoxin reductase and three heat-labile proteins (designated PI, PII, and PIII) that have thioredoxin- or glutaredoxin-like properties are found. Both PI and PIII have molecular masses of approximately 12,000 daltons and are readily reduced by their homologous HeLa thioredoxin reductase. However, only PI can be reduced efficiently by the glutathione system and neither PI nor PIII has inherent glutathione-disulfide oxidoreductase activity. PII has a molecular mass of greater than 30,000 daltons and appears to be associated with a reductase activity. The HeLa NADPH-thioredoxin reductase has been purified to near homogeneity and found to be a 116,000-dalton flavoprotein composed of two 58,000-dalton subunits. The HeLa enzyme has low species and substrate specificity and can reduce HeLa PI and PIII, E. coli thioredoxin and glutaredoxin, and the disulfide bond in 5,5'-dithiobis(2-nitrobenzoic acid). The exact in vivo roles of the HeLa thioredoxin/glutaredoxin system remain to be determined.

Thioredoxin/Glutaredoxin System of Chlorella: CHLORELLA ADENOSINE 5'-PHOSPHOSULFATE SULFOTRANSFERASE CANNOT USE THIOREDOXIN OR GLUTAREDOXIN AS COFACTORS.

Using the thioredoxin/glutaredoxin-dependent adenosine 3'-phosphate 5'-phosphosulfate reductase coupled assay system, the Chlorella thioredoxin/glutaredoxin system has been partially purified and characterized. A NADPH-thioredoxin reductase and two thioredoxin/glutaredoxin activities, designated as Chlorella thioredoxin/glutaredoxin protein I and II (CPI and CPII), were found in crude extracts of Chlorella. Similar to their counterparts from Escherichia coli, both CPI and CPII are heat-stable low molecular proteins of approximately 14,000. While CPI (but not CPII) is a substrate for its homologous NADPH-thioredoxin reductase as well as for E. coli NADPH-thioredoxin reductase, CPII is better than CPI as a substrate for reduction by the glutathione system. Based on these properties, CPI and CPII may be classified as Chlorella thioredoxin and Chlorella glutaredoxin, respectively. The Chlorella NADPH-thioredoxin reductase (M(r) = 72,000, with two 36,000-dalton subunits) resembles E. coli-thioredoxin reductase in size. Besides Chlorella thioredoxin, the Chlorella thioredoxin reductase will also use E. coli thioredoxin, but not glutaredoxin, as a substrate. Although a thioredoxin-like protein has been implicated in higher plant light-dependent sulfate reaction, neither Chlorella thioredoxin nor glutaredoxin can stimulate the thiol-dependent adenosine 5'-phosphosulfate-sulfotransferase reaction. Furthermore, Chlorella thioredoxin and glutaredoxin, in conjunction with an appropriate reductase system, cannot replace the thiol requirement of Chlorella adenosine 5'-phosphosulfate-sulfotransferase. The exact physiological roles and subcellular localization of the Chlorella thioredoxin and glutaredoxin systems remain to be determined.